Mark A. Carlson, MD

Department of SurgeryUniversity of Nebraska Medical Center

Omaha VA Medical CenterOmaha, Nebraska

USA

Skin Regeneration

UNMC Dept Surgery Grand Rounds, July 14, 2010

Disclosures:none.

Content available: www.nebraskasurgicalresearch.com

Overview

• Clinical problem• Established treatments• Research approaches• Our strategy

Skin Loss:Clinical Problem

• Normal anatomy• Healing v. Regeneration• Sequelae of skin loss

Normal skin anatomy (rat)

1 mm

dermis

panniculuscarnosus

epidermis

5 µm

100 µm

dermis

epidermis

cf

fb

cf = collagen fibrilfb = fibroblast

subcutis

Normal skin anatomy

structural components functions

• epidermis• basal lamina• dermis• blood vessels• hair follicles• glands• cutis (nail, claw, horn)• (subcutaneous adipose)• (skeletal muscle)

• barrier• temperature regulation• sensation• coloration• immune response• synthetic• specialized

Healing vs. Regeneration

Excisional wound: natural history

Dorsum of rat: 2 x 2 cm full-thickness excision,followed for 204 days

1 cm

Growth(RJ Goss, 1992)

intact organism

compensatoryhypertrophy

tissue loss

pathologicprocess wound healing

EPIMORPHIC REGENERATION

Growth Redux

Healing v. Regeneration

Amphibian limb amputation

A type of growth: wound healing (corneal injury)

Regeneration vs. conventional healing

Elastin: scar vs. dermis scar

Negative growth:

atrophy

(skeletal muscledenervation)

rat tibialis anterior(anti-laminin)

control

1 mo

2 mo

4 mo

(Anat Rec 2001;264:203)

tissue loss/injury

response

death

recovery

inflammation regeneration

healing

scar with minor disability

quality of life scalepoor good

scar with major disability

Paradigm

organism size

regenerativeability

organism complexity

Regeneration through the phyla

The larger and/or more advanced theanimal, the less it can regenerate.

Hydra

H. simpson

HIGH

1. Epidermis2. Liver (hyperplasia)3. Endothelium4. Epithelium

LOW (but theoretically possible)

1. Everything else

Tissue-Specific Mammalian Regenerative Capacity

Holy Grail

So what is the problem with healing?

In most cases, nothing. But…

Sequela of skinloss:

Burn woundcontracture

Abnormalhealing

response:

Keloidscarring

“Tissue repair is designed to reconstruct morphological integrity;epimorphic regeneration is designed to restore function.”

“Animals can live without epimorphic regeneration but notwithout tissue repair.”

Richard J. Goss, 1992

Wound healing = quick fix; poor function

Regeneration = complicated; normal function

Skin Loss:Established Treatments

• Skin flaps/grafts• Epidermal substitutes• Dermal substitutes• Combined (epiderm + derm)

Skin flaps:simple

Excisional wound(rat dorsum)

Free flap

(treatment ofcontracture)

Skin grafting

Harvest(300-400 µm thick)

Forearm graft

Skin grafting:

full- vs. split-thickness

split-thickness

Skin grafting:

Skin grafting:

“Gold Standard” for skin replacement

Disadvantages

• Limited source of material• Wound contraction• Cosmesis• Donor site morbidity

Skin grafting:Donor site morbidity

Skin loss treatments:Epidermal substitutes

• Strategy: cultured autologouskeratinocytes/hair follicle cells (CEA)

• Examples: Epicel®, Laserskin®,Epidex™, MySkin™

Epidermal replacement

Epidermal substitutes:Autologous keratinocytes

Advantages:• No rejection• No large donor sites

Disadvantages:• Fragile, no supportive dermis• Time requirement (culture), no storage

Epicel

Skin loss treatments:Dermal substitutes

• Strategy: natural, semisynthetic, orsynthetic matrix, ± fibroblasts

• Examples: Alloderm®, Biobrane®,Dermagraft®, Integra®,Permacol™, Transcyte®

Dermal replacement

Dermal substitute:Alloderm®

Composition: processed cadaveric humandermis

Advantages• Minimal rejection• Simplicity, tractabilityDisadvantages• No cellular component• Expensive

ePlasty.com

Dermal substitute:Biobrane®

Composition: nylon mesh + siliconemembrane + porcine ECM

Advantages• Simplicity• Large area coverageDisadvantages• Not a permanent replacement

burn wound with Biobrane

Dermal substitute:Dermagraft®

Composition: allogeneic neonatalfibroblasts in polyglactin mesh

Advantages• Minimal rejection• Absorbable ECMDisadvantages• Complexity• Small area coverage

Dermal substitute:Integra®

Composition: bovine col + GAGs topped withsilicone

Advantages• Encourage cellular ingrowth• Integrates with hostDisadvantages• Needs autograft after silicone removal• Bovine allergy risk

Dermal substitute:Permacol™

Composition: treated porcine dermal collagenAdvantages• Non-immunogenic• Supports ingrowth from hostDisadvantages• Needs autograft after incorporation• Expensive

Dermal substitute:Transcyte®

Composition: collagen-coatednylon seeded with allogeneicneonatal fibroblasts, toppedwith nylon

Advantages• Integrates with host, encourages ingrowthDisadvantages• Nonabsorbable• Not permanent

Skin loss treatments:Combined epidermal/dermal substitutes

(composites)

• Strategy: cultured allogeneic cellspopulating a bilaminar structure

• Examples: Apligraf®, Orcel®

Composite substitute:Apligraf®; Orcel®

Composition: allogeneic neonatal keratinocytes +fibroblasts, type I col matrix with cytokines

Advantages• Minimal rejection• Rational designDisadvantages• Complexity, time, expense• Limited area coverage

Apligraf

Orcel

Skin Regeneration:Research Approaches

• Endogenous regeneration

• Fetal paradigm

• Tissue engineering

Research approaches:Endogenous regeneration

• Enable inherent regenerative mechanism(“inside-out” approach)

• Requires understanding of regenerationvs. healing

• Resident stem cell biology relevant

“Mammals do not regenerate”

Exceptions:

• Antler growth

• Rabbit ear regeneration

• Distal fingertip amputations in miceand young children

(Others?)

Blastema formation:central event of limb regeneration

(Goss, 1992)

Dedifferentiation in nature?

Limb amputation blastema

?

What’s in there?

A. Stem cells?B. Dediff cells?C. A & B?

Dedifferentiation in nature?

Stem Cells:Properties

1. Self-renewal (can make a copy of itself)

2. Differentiation (can become another cell type)

3. Can reconstitute tissue in vivo [ informal ]

blastocyst ESC colony

Stem Cell Primer

• ESC: embryonic stem cell (blastocyst ICM)

• iPS: induced pluripotent stem cell

• Tissue stem cell (adult or somatic stem cell)

• MMSC: multipotent mesenchymal stromal cell

blastocyst ESC colony

Stem cell potency

Totipotent Pluripotent Multipotent Progenitor

zygote embryonic stem hematopoietic stem intestinal crypt

MesodermEndodermEctoderm

and

GermTrophoblast

MesodermEndodermEctoderm

(i.e., entire organism)

Organ specific Cell specific

PLASTICITY

Unipotent/

undifferentiated stem cell

differentiated daughter cell

forward pathway: canonical(differentiation)

reverse pathway =DEDIFFERENTIATION

(Der Alchemist, E. van Hove)

Stem cells & dedifferentiation

• Does this pathway exist?• If so, where, how, and how

frequently?

Research approaches:Fetal paradigm

• Mammalian fetus: regenerative responseinstead of inflammation/healing

• Transition point in utero, after whichregeneration no longer occurs

• Phenomenology followed by mechanisticstudies (no applications yet)

E16 E18

Excisional wounds in the fetal rat after 72 hr

Scarless healing in the dermis of the mammalian fetus

(Plast Reconstr Surg 2001;160:209)

72 hr after wounding on E16 unwounded

Scarless fetal healing (cont’d):collagen organization in the E16 fetus

72 hr after wounding on E18 unwounded

Scarless fetal healing (cont’d):collagen organization in the E18 fetus

Fetal paradigm:Comparison with adult

• Fetal ECM: enriched in type III collagen,hyaluronic acid, tenascin-C

• Elevated levels of TGF-β3, IL-10;decreased TGF-β1/2, IL-8, many others

• Differences in fibroblast phenotype

Research approaches:Tissue engineering

• Replace lost tissue with engineeredconstruct (“outside-in” approach)

• Requires understanding of how implantsinteract with host

• Pluripotent stem cell biology relevant

Tissue engineering:scaffolds for construction

• Materials include organic polymers (e.g.,polyglactin, polylactide), gelatin, chitosan,PEG hydrogels

• A variety of synthetic techniques cancontrol micro- and nano-architecture (e.g.,fiber diameter, pore size, physicalproperties)

Tissue engineering:“Smart” biomaterials

• Older strategy of 100% synthetic matrixfailed

• Newer strategy uses semisyntheticmaterials (e.g., polymer coated with ECM)

• Coating encourages cellular ingrowth

Tissue engineering:ECM coating

• Adsorbed or covalently bound to scaffold

• Examples include: type I/III collagens,elastin, HA, fibronectin, RGD peptides,other peptide mimetics

Tissue engineering:cellular embedding

• Embed vs. ingrowth (chemoattraction)

• Cell type: differentiated vs. stem

• If stem, what type (MSC, iPSC, ESC, etc.)

Skin Regeneration:Our Strategy

• Nanoengineered scaffold• ECM surfacing• Cells (MSC + fibroblast)• Cytokine slow-release• Multi-layer recapitulation

Objective:

To develop a replacement therapy forepidermis/dermis (i.e., to engineer a

complete skin equivalent for clinical use)

dermal replacement: strategy

synthetic backbone

20 µm 100 µm 20 µm

• Material can be manipulated atthe nano-level for architecturaland physical properties

• Material can be engineered tocontain nanoparticles for slow-release of various cytokines/growth factors

collagen matrix with cells:• dermal fibroblasts• mesenchymal “stem”

5 mm

50 µm 10 µm

dermal replacement: strategy

“neodermis”

dermal replacement: strategy

basal lamina(type IV collagen, laminin)

neodermis

dermal replacement: strategy

keratinocytes(epidermis)

dermal replacement: strategy

plug intoanimal model

finished skin equivalent

Aerosol Delivery System

[to go movie file “ADS.mov”]

“Regeneration” Assay

(wound contraction)PWD 0

PWD 0

PWD 7

PWD 14

PWD 28

PWD 204

1 cm

0

1

2

3

4

0 60 120 180w

ound a

rea (

cm

2)

PWD

goal

Additional endpoints• Tensile strength• Microscopic morphology

Looks easy, but … potential problems:

• Co-culture conditions• Blood supply to implant• Infection (see next item)• Inflammation at interface• Strength & durability• Nerves, glands, hair, etc.• Translation from rodents to humans• Cellular source

Engineered tissue will require an immediate blood supply

(Donor kidney just prior to transplantation)

And…

Cell 2003;114:763 red = cardiac myosingreen = adult stem cells, derived from myocardiumblue = nucleimagenta = α-smooth muscle actin

rat LV

infarct

…Engineered tissue may need to function immediately after implantation.

Inflammation

(Excisional wound bed, postwounding day 3)

Conclusions1. Proofs of concept are readily

available, but…

2. Translation into practicaltreatments have been rare

3. Road to practical treatmentswill be long and difficult

Current reality:

“When my liver fails, don't give me a bone marrowtransplant [i.e., stem cells], give me a liver.”

Irving Weissman, 2004

Take-home message:

Healing bad, regeneration good.

(end)